Tablet formulation of ezatiostat

ABSTRACT

Disclosed herein are tablets comprising ezatiostat hydrochloride wherein the ezatiostat hydrochloride comprises from about 75 to about 82 percent by weight of the tablet.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit under 35 USC 119(e) of U.S.Provisional Application No. 61/352,377, filed on Jun. 7, 2010, thecontents of which are incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

This invention relates to tablets comprising ezatiostat hydrochloride.

STATE OF THE ART

Ezatiostat and its salts are disclosed in U.S. Pat. No. 5,763,570.Ezatiostat has the IUPAC chemical name of ethyl(2S)-2-amino-5-[[(2R)-3-benzylsulfanyl-1-[[(1R)-2-ethoxy-2-oxo-1-phenylethyl]amino]-1-oxopropan-2-yl]amino]-5-oxopentanoate.

It has been discovered that ezatiostat salts and, in particular, thehydrochloride salt, can be formed as a crystalline ansolvate, referredto as form D, which is disclosed in U.S. application Ser. No.13/041,136, the contents of which are incorporated herein by referencein its entirety.

Ezatiostat hydrochloride (USAN) has the molecular weight of 566.1, thetrademark of Telintra®, and the CAS registry number of 286942-97-0.Ezatiostat hydrochloride has been evaluated for the treatment ofmyelodysplastic syndrome (MDS), in a Phase I-IIa study using a liposomalformulation (U.S. Pat. No. 7,029,695), as reported at the 2005 AnnualMeeting of the American Society for Hematology (Abstract #2250) and byRaza et al. in Journal of Hematology & Oncology, 2:20 (published onlineon 13 May 2009); and in a Phase I study using a tablet formulation, asreported at the 2007 Annual Meeting of the American Society forHematology (Abstract #1454) and by Raza et al. in Blood, 113:6533-6540(prepublished online on 27 Apr. 2009), and in a single patient casereport by Quddus et al. in Journal of Hematology & Oncology, 3:16(published online on 23 Apr. 2010). The entire disclosures of each ofthe patents and publications referred to in this application areincorporated into this application by reference.

The clinical tablet formulation of ezatiostat hydrochloride fortreatment of MDS employs tablets containing 500 mg of ezatiostathydrochloride. When so employed, tablet size and its ability to beswallowed by patients who are typically elderly becomes a problem.Typically, tablets employ a variety of pharmaceutically acceptableexcipients which can range up to 90+ weight percent of the total weightof the tablet. When the excipients' content is so high, there is lessconcern about sizing the tablet as adjusting the amount of excipientscan reduce the weight of the tablet and, accordingly, its size.

In the case of ezatiostat hydrochloride with its large molecular weightand the required amount of actives, tablets sized at a level suitablefor oral delivery to human patients, especially elderly patients, mustcomprise from about 75 to 82 weight percent of that drug. This, in turn,imparts significant difficulty in preparing suitable tablets. Forexample, tablets containing this much of the active drug, in addition tobeing suitably sized, must meet pharmaceutical characteristics whichinclude among others, flow characteristic, granulate density, granulatecompressibility, suitable integrity during manufacture, shipping andstorage, proper shelf-life, and suitable disintegration properties wheningested. Given the weight percent of ezatiostat hydrochloride in thesetablets, the amount of excipients used to make a pharmaceutically usefultablet is strictly limited.

Accordingly, while there is a need for tablets of ezatiostathydrochloride which permit a suitably sized tablet containing from about75 to about 82 weight percent of drug, the ability of forming a tabletwith that much active drug is exceptionally problematic.

SUMMARY OF THE INVENTION

This invention is directed to the surprising and unexpected discoverythat pharmaceutically acceptable tablets of ezatiostat hydrochloride canbe prepared using 75 to 82 weight percent of the drug while stillmaintaining all of the properties required of a tablet.

Accordingly, in one embodiment, this invention is directed topharmaceutically acceptable tablets comprising ezatiostat hydrochloride,an intragranular excipient, and an extragranular excipient, wherein theezatiostat hydrochloride comprises from about 75 to about 82 percent byweight of the tablet.

In another embodiment of this invention, the tablets contain from about100 mg to about 1250 mg ezatiostat hydrochloride and employ one or moreintragranular excipients and one or more extragranular excipients.

In another embodiment of this invention, the intragranular excipientcomprises one or more of mannitol, croscarmellose sodium, hypromellose.In another embodiment, the intragranular excipient comprises a mixtureof each of these components. The total amount of the intragranularexcipient is from about 17 to about 21 weight/weight percent based onthe total weight of the tablet and preferably from about 19 to about 20weight/weight percent.

In a preferred embodiment, the amount of mannitol employed in theintragranular excipient mixture ranges from about 13 to about 15weight/weight percent and preferably from about 13.5 to about 14.5weight/weight percent based on the total weight of the tablet. Themannitol acts as a diluent in the intragranular agglomerate.

In a preferred embodiment, the amount of croscarmellose sodium employedin the intragranular excipient mixture ranges from about 1.5 to about3.5 weight percent and preferably from about 2 to about 3 weight percentbased on the total weight of the tablet. The croscarmellose sodium actsas a disintegrant in the intragranular agglomerate.

In a preferred embodiment, the amount of hypromellose employed in theintragranular excipient mixture ranges from about 2 to 4 weight percentand preferably from about 2.5 to about 3.5 weight percent based on thetotal weight of the tablet. The hypromellose acts as a binder in theintragranular agglomerate.

As per the examples below, the intragranular excipient mixture isblended with the drug to provide a cohesive agglomerate. Thisagglomerate is formed into granules which are then combined with anextragranular excipient mixture, blended and formed into tablets. In oneembodiment, the extragranular excipients include magnesium stearate andcroscarmellose sodium.

In a preferred embodiment, the amount of croscarmellose sodium employedin the extragranular excipient mixture ranges from about 1 to 4 weightpercent and preferably about 1.5 to about 3.5 weight/weight percent andeven more preferably about 2 to about 3 weight/weight percent based onthe total weight of the tablet. The croscarmellose sodium acts as adisintegrant in the formed tablet.

In a preferred embodiment, the amount of magnesium stearate employed inthe extragranular excipient mixture ranges from about 0.5 to about 1.5weight/weight percent and preferably about 1 weight/weight percent basedon the total weight of the tablet. The magnesium stearate acts as alubricant in the formed tablet.

As used above, the total weight of the tablet is based on the amount ofdrug, the amount of intragranular excipients and the amount ofextragranular excipients. The amount of water in the tablet isnegligible. Optionally, a coating can be applied to the tablets. Anysuch coating is not included in the weight of the tablet for thepurposes of determining the weight percentages recited herein. Theoptional coating includes pharmaceutically acceptable coatingexcipients. Preferably, such excipients include a combination ofpolyethylene glycol and hypromellose as coating agents.

As provided in the examples below, it has been surprisingly found thatthe tablets of this invention are pharmaceutically acceptablenotwithstanding the very high percentage of drug employed in thesetablets.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 is a DSC pattern of ezatiostat hydrochloride monohydrate form A.

FIG. 2 is a DSC pattern of crystalline ezatiostat hydrochlorideansolvate form D.

FIG. 3 is an XRPD pattern of crystalline ezatiostat hydrochlorideansolvate form D.

FIG. 4 is a high-resolution XRPD pattern of crystalline ezatiostathydrochloride ansolvate form D.

FIG. 5 is an SS-NMR spectrum of crystalline ezatiostat hydrochlorideansolvate form D.

FIG. 6 is a comparative DSC pattern of crystalline ezatiostathydrochloride polymorphic forms A, D, and E.

DETAILED DESCRIPTION

This invention is directed to tablets comprising ezatiostathydrochloride. However, prior to describing this invention in moredetail, the following terms will first be defined.

As used herein, the term “comprising” or “comprises” is intended to meanthat the compositions and methods include the recited elements, but notexcluding others. “Consisting essentially of” when used to definecompositions and methods, shall mean excluding other elements of anyessential significance to the combination for the stated purpose. Thus,a composition consisting essentially of the elements as defined hereinwould not exclude other materials or steps that do not materially affectthe basic and novel characteristic(s) of the claimed invention.“Consisting of” shall mean excluding more than trace elements of otheringredients and substantial method steps. Embodiments defined by each ofthese transition terms are within the scope of this invention.

The term “about” when used before a numerical designation, e.g.,temperature, time, amount, and concentration, including range, indicatesapproximations which may vary by (+) or (−) 15%, 10%, 5% or 1%.

The singular forms “a,” “an,” and “the” and the like include pluralreferents unless the context clearly dictates otherwise. Thus, forexample, reference to “a compound” includes both a single compound and aplurality of different compounds.

The “crystalline ansolvate” of ezatiostat hydrochloride is a crystallinesolid form of ezatiostat hydrochloride, such as, e.g., the crystallineform D. The form D crystal lattice is substantially free of solvents ofcrystallization. However, any solvent present is not included in thecrystal lattice and is randomly distributed outside the crystal lattice.Therefore, form D crystals in bulk may contain, outside the crystallattice, small amounts of one or more solvents, such as the solventsused in its synthesis or crystallization. As used above, “substantiallyfree of” and “small amounts,” refers to the presence of solventspreferably less than 10,000 parts per million (ppm), or more preferably,less than 500 ppm.

“Characterization” refers to obtaining data which may be used toidentify a solid form of a compound, for example, to identify whetherthe solid form is amorphous or crystalline and whether it is unsolvatedor solvated. The process by which solid forms are characterized involvesanalyzing data collected on the polymorphic forms so as to allow one ofordinary skill in the art to distinguish one solid form from other solidforms containing the same material. Chemical identity of solid forms canoften be determined with solution-state techniques such as ¹³C NMR or ¹HNMR. While these may help identify a material, and a solvent moleculefor a solvate, such solution-state techniques themselves may not provideinformation about the solid state. There are, however, solid-stateanalytical techniques that can be used to provide information aboutsolid-state structure and differentiate among polymorphic solid forms,such as single crystal X-ray diffraction, X-ray powder diffraction(XRPD), solid state nuclear magnetic resonance (SS-NMR), and infraredand Raman spectroscopy, and thermal techniques such as differentialscanning calorimetry (DSC), thermogravimetry (TG), melting point, andhot stage microscopy.

To “characterize” a solid form of a compound, one may, for example,collect XRPD data on solid forms of the compound and compare the XRPDpeaks of the forms. For example, when only two solid forms, I and II,are compared and the form I pattern shows a peak at an angle where nopeaks appear in the form II pattern, then that peak, for that compound,distinguishes form I from form II and further acts to characterize formI. The collection of peaks which distinguish form I from the other knownforms is a collection of peaks which may be used to characterize form I.Those of ordinary skill in the art will recognize that there are oftenmultiple ways, including multiple ways using the same analyticaltechnique, to characterize solid forms. Additional peaks could also beused, but are not necessary, to characterize the form up to andincluding an entire diffraction pattern. Although all the peaks withinan entire XRPD pattern may be used to characterize such a form, a subsetof that data may, and typically is, used to characterize the form.

An XRPD pattern is an x-y graph with diffraction angle (typically ° 2θ)on the x-axis and intensity on the y-axis. The peaks within this patternmay be used to characterize a crystalline solid form. As with any datameasurement, there is variability in XRPD data. The data are oftenrepresented solely by the diffraction angle of the peaks rather thanincluding the intensity of the peaks because peak intensity can beparticularly sensitive to sample preparation (for example, particlesize, moisture content, solvent content, and preferred orientationeffects influence the sensitivity), so samples of the same materialprepared under different conditions may yield slightly differentpatterns; this variability is usually greater than the variability indiffraction angles. Diffraction angle variability may also be sensitiveto sample preparation. Other sources of variability come from instrumentparameters and processing of the raw X-ray data: different X-rayinstruments operate using different parameters and these may lead toslightly different XRPD patterns from the same solid form, and similarlydifferent software packages process X-ray data differently and this alsoleads to variability. These and other sources of variability are knownto those of ordinary skill in the pharmaceutical arts. Due to suchsources of variability, it is usual to assign a variability of ±0.2° 2θto diffraction angles in XRPD patterns.

X-ray powder diffraction (XRPD) analyses were performed on a ShimadzuXRD-6000 X-ray powder diffractometer using Cu Kα radiation from a longfine focus X-ray tube, operated at 40 kV, 40 mA. The divergence andscattering slits were set at 1° and the receiving slit was set at 0.15mm. Diffracted radiation was detected by a NaI scintillation detector. A0-2θ continuous scan at 3°/min (0.4 sec/0.02° step) from 2.5°-40° 2θ wasused. A silicon standard was analyzed to check alignment of theinstrument. Data were collected and analyzed using XRD-6000 v. 4.1software.

High-resolution XRPD analyses were also performed on a PANalyticalX'Pert PRO PW3040 diffractometer, using Cu Kα radiation produced by anOptix long fine-focus tube (45 kV, 40 mA). An elliptically gradedmultilayer mirror was used to focus the X-rays through the specimen,which was sandwiched between 3 μm films, analyzed in transmissiongeometry, and rotated to optimize orientation statistics. A beam-stopand helium purge were used to minimize the air-scattering background;Soller slits (divergence slit, 0.5°; scattering slit 0.25°) were usedfor the incident and diffracted beams to minimize axial divergence.Diffraction patterns were collected using a scanning position-sensitiveX'Celerator detector located 240 mm from the specimen, over a scan rangeof 1.01°-39° 2θ with a scan speed of 1.2°/min (step size 0.017° 2θ). Asilicon standard was analyzed to check alignment of the instrument. Datawere collected and analyzed using X'Pert PRO Data Collector v. 2.2bsoftware. Indexing and Pawley refinement of the ezatiostat hydrochloridemonohydrate XRPD pattern was performed using Match v.2.4.0 software(SSCI) and verified using ChekCell v. Nov. 1, 2004. Indexing and Pawleyrefinement of the crystalline ezatiostat hydrochloride ansolvate XRPDpattern was performed using DASH v. 3.1 software (CambridgeCrystallographic Data Center).

Variable-temperature XRPD (VT-XRPD) analysis was performed on a ShimadzuXRD-6000 diffractometer equipped with an Anton Paar HTK 1200 hightemperature stage. The sample was packed in a ceramic holder andanalyzed from 2.5°-40° 2θ at 3°/min (0.4 sec/0.02° step). Thetemperature was held constant during each XRPD scan. Temperaturecalibration was performed using vanillin and sulfapyridine standards. Asilicon standard was analyzed to check alignment of the instrument; datawere collected and analyzed using XRD-6000 v. 4.1 software.

Differential scanning calorimetry (DSC) analyses were performed on a TAInstruments Q100 or 2920 differential scanning calorimeter, which wascalibrated using indium as the reference material. The sample was placedinto a standard aluminum DSC pan with an uncrimped lid, and the weightaccurately recorded. The sample cell was equilibrated at 25° C. andheated under a nitrogen purge at a rate of 10° C./minute to a finaltemperature of 250° C. The variability of DSC data is affected by samplepreparation and particularly by heating rate.

Solid-state NMR(SS-NMR) ¹³C cross-polarization magic angle spinning(CP/MAS) analyses were performed at room temperature on a Varian^(UNITY) INOVA-400 spectrometer (Larmor frequencies: ¹³C=100.542 MHz,¹H=399.800 MHz). The sample was packed into a 4 mm PENCIL type zirconiarotor and rotated at 12 kHz at the magic angle. The spectrum wasacquired with phase modulated SPINAL-64 high power ¹H decoupling duringthe acquisition time using a ¹H pulse width of 2.2 μs (90°), a rampedamplitude cross polarization contact time of 2 ms, a 30 ms acquisitiontime, a 5 second delay between scans, a spectral width of 45 KHz with2700 data points, and 200 co-added scans. The free induction decay (FID)was processed using Varian VNMR 6.1C software with 32768 points and anexponential line broadening factor of 10 Hz to improve thesignal-to-noise ratio. The first three data points of the FID were backpredicted using the VNMR linear prediction algorithm to produce a flatbaseline. The chemical shifts of the spectral peaks were externallyreferenced to the carbonyl carbon resonance of glycine at 176.5 ppm. Thevariability of SS-NMR peaks in this experiment is considered to be ±0.2ppm.

Karl Fischer analyses for water determination were performed on aMettler Toledo DL39 Karl Fischer titrator. About 10-15 mg of sample wasplaced in the KF titration vessel containing approximately 100 mL ofHydranal®—Coulomat AD reagent and mixed for 60 seconds to ensuredissolution. The dissolved sample was then titrated by means of agenerator electrode which produces iodine by electrochemical oxidation.

Thermogravimetric (TG-IR) analyses were performed on a TA Instrumentsmodel 2050 thermogravimetric (TG) analyzer interfaced to a ThermoNicolet Magna® 560 Fourier transform infrared (FT-IR) spectrophotometerequipped with a Ever-Glo mid/far IR source, a potassium bromidebeamsplitter, and a deuterated triglycine sulfate detector. Theinstrument was operated under a flow of helium at 90 mL/min (purge) and10 mL/min (balance). The sample was placed in a platinum sample pan,inserted into the TG furnace, accurately weighed by the instrument, andheated from ambient at a rate of 20° C./min. The TG instrument wasstarted first, immediately followed by the FT-IR instrument. IR spectrawere collected every 12.86 seconds; and each IR spectrum represents 32co-added scans collected at a spectral resolution of 4 cm⁻¹. Abackground scan was collected before the beginning of the experiment.Wavelength calibration was performed using polystyrene. The TGcalibration standards were nickel and Alumel™.

Hot stage microscopy analysis was performed on a Linkam FTIR 600 hotstage mounted on a Leica DM LP microscope. Samples were observed using a20× objective with cross polarizers and lambda compensator. A coverslipwas then placed over the sample. Each sample was visually observed asthe stage was heated. Images were captured using a SPOT Insight™ colordigital camera with SPOT Software v. 3.5.8. The hot stage was calibratedusing USP melting point standards.

The term “does not undergo polymorphic transformation” refers to noobservable polymorphic transformation of a crystalline form, whenexposed to up to about 75% relative humidity at up to about 40° C. forup to about 6 months, when analyzed by XRPD or HPLC or anotherequivalently sensitive technique.

“Desiccant” refers to a substance that induces or sustains a state ofdryness in its local vicinity in a moderately well-sealed container.Desiccants can absorb or adsorb water, or act by a combination of thetwo. Desiccants may also work by other principles, such as chemicalbonding of water molecules. A pre-packaged desiccant may be used toremove excessive humidity that would degrade products. Non-limitingexamples of desiccants include silica gel, calcium sulfate, calciumchloride, montmorillonite clay, and molecular sieves.

“Room temperature” refers to (22±5)° C.

“Storing” or “storage” refers to storing crystalline ezatiostathydrochloride. In a preferred embodiment, crystalline ezatiostathydrochloride comprises ansolvate form D or a composition including theform D such that no more than about 10%, more preferably no more thanabout 5%, still more preferably no more than about 3%, or mostpreferably no more than about 1% of the ansolvate form D undergoestransformation to another compound.

METHODS OF THE INVENTION Tablet Preparation

Ezatiostat hydrochloride tablets prepared below are preferably modifiedcapsule shaped, coated tablets containing 500 mg of ezatiostathydrochloride.

The ezatiostat hydrochloride used in the tablets may be in hydrated orin crystalline anhydrous form (though the weights and weight percentagesin the specification and claims are based on anhydrous ezatiostathydrochloride).

A crystalline anhydrous form of ezatiostat hydrochloride may be obtainedby heating hydrated ezatiostat hydrochloride to temperatures over about125° C. (the temperature required being dependent on the initial levelof hydration: about 130° C. for a polyhydrate containing approximately 5molecules of water per molecule of ezatiostat hydrochloride to about153° C. for ezatiostat hydrochloride monohydrate), or may be obtained byslurrying hydrated ezatiostat hydrochloride in methyl tert-butyl etherat ambient temperature or in hexanes at elevated temperatures such as60° C. If the ezatiostat hydrochloride is crystallized at the end of itssynthesis, crystalline anhydrous ezatiostat hydrochloride may beobtained by dissolution of crude hydrated ezatiostat hydrochloride inabout 5.6 times its weight of ethanol, heating to about 65°-70° C.,filtering, seeding with a small quantity (e.g. about 2% by weight of theinitial ezatiostat hydrochloride) of crystalline anhydrous ezatiostathydrochloride, cooling to about 40° C., adding ethyl acetate in about13.5 times the weight of the ezatiostat hydrochloride, gradually coolingto about 20°-25° C. and then to −5°-0° C., then filtering, washing withethyl acetate, and drying. This crystalline anhydrous form has a meltingpoint of about 166° C., and is characterized by an orthorhombic spacegroup (P2₁2₁2 or P2₁2₁2₁) with approximate unit cell dimensions ofα=64.2±0.2 Å, b=18.3±0.1 Å, c=5.1±0.1 Å

Each tablet contains ezatiostat hydrochloride in a formulationcontaining mannitol, croscarmellose sodium, hypromellose, magnesiumstearate, and optionally coated with mixture of hypromellose andpolyethylene glycol 400 (also referred to as Opadry® Clear). Purifiedwater, used during granulation and coating, is removed duringprocessing. The quantitative composition of ezatiostat hydrochloridetablets is provided in Table 1.

TABLE 1 Quantitative Composition of Ezatiostat Hydrochloride TabletsFinal Amount per Composition Tablet (mg) Ingredient and Grade Percent(w/w) 500 mg Tablet Drug Substance Ezatiostat hydrochloride 76.9  500Intragranular Excipients Mannitol, USP 14.1  91.5 Croscarmellose Sodium,NF 2.5 16.23 Hypromellose, USP 3.0 19.5 Purified Water, USPNegligible^(a) Negligible^(a) Extragranular Excipients CroscarmelloseSodium, NF 2.5 16.3 Magnesium Stearate, NF 1.0 6.5 Core Tablet Total100   650 Optional Coating Excipients Hypromellose, USP 3^(b ) 19.5Polyethylene Glycol 400, NF Purified Water, USP Negligible^(a)Negligible^(a) Coated Tablet Total NA 670 ^(a)Water is removed duringprocessing. ^(b)3% w/w of combined coating excipients. NA = notapplicable.

The inactive ingredients in ezatiostat hydrochloride tablets and thegrade of those ingredients are listed in Table 2. The rationale for eachof the ingredients is also provided. For the intragranular excipients,mannitol functions as a diluent, croscarmellose sodium as adisintegrant, hypromellose as a binder and purified water as thegranulation fluid. Extragranular excipients include croscarmellosesodium as a disintegrant and magnesium stearate as a lubricant. Coatingagents consist of hypromellose and polyethylene glycol 400. These twocombined excipients are used as a product called Opadry® Clear.

The inactive ingredients conform to current compendial monographrequirements according to the USP and National Formulary (NF) and areprovided in Table 2 below.

TABLE 2 Inactive Ingredients in Ezatiostat HCl Tablets InactiveIngredients Grade Function Intragranular Excipients Mannitol USP DiluentCroscarmellose Sodium NF Disintegrant Hypromellose^(a) USP BinderPurified Water^(b) USP Granulation Fluid Extragranular ExcipientsCroscarmellose Sodium NF Disintegrant Magnesium Stearate NF LubricantCoating Excipients Hypromellose^(a,c) USP Coating Agent PolyethyleneGlycol 400^(c) NF Coating Agent Purified Water^(b) USP Solvent^(a)Hypromellose is also known as hydroxypropyl methylcellulose (HPMC).^(b)Water is removed during processing. ^(c)Hypromellose andPolyethylene Glycol 400 are the ingredients in Opadry ® Clear.

Ezatiostat hydrochloride tablets are manufactured by dry blendingezatiostat hydrochloride and the intragranular excipients, adding waterand wet screening the granulate. The granulate is then dried in a fluidbed dryer and the dried granules are milled. The dried, milled granulesare blended with the extragranular excipients in a V blender. After theblend is compressed into tablets, they are film coated with Opadry®Clear solution. The general manufacturing process for the tablets isdiscussed in Example 2.

Ezatiostat hydrochloride tablets are packed in white high densitypolypropylene (HDPE) bottles, with a white HDPE outer cap andpolypropylene (PP) inner cap over an induction seal. Each bottlecontains either 50 tablets or 150 tables for 500 mg strength. A smallcanister containing silica gel desiccant has been placed in the tabletbottles to reduce moisture and improve stability (shelf-life).

Ezatiostat hydrochloride tablets can be stored at 2° C.-30° C. Thetablets of this invention have a shelf-life of at least 48 months whenstored with a desiccant at 25° C. and 60% relative humidity. When soprepared, the tablets of this invention are suitable for pharmaceuticaluse including among others a suitable size and possess acceptablehardness, dissolution and shelf-life.

In one embodiment, this invention provides a pharmaceutically acceptabletablet comprising ezatiostat hydrochloride, an intragranular excipientand extragranular excipient wherein the ezatiostat hydrochloridecomprises from about 75 to about 82 percent by weight of the tablet,said tablet further comprises a film coating.

In another embodiment, this invention provides a pharmaceuticallyacceptable tablet comprising ezatiostat hydrochloride, an intragranularexcipient and extragranular excipient wherein the ezatiostathydrochloride comprises from about 75 to about 82 percent by weight ofthe tablet, wherein said tablet comprises about 500 mg of ezatiostathydrochloride. In another embodiment, the tablet comprises preferablyabout 750 mg, more preferably about 1 gm, and even more preferably about1.25 gm of ezatiostat hydrochloride.

In another embodiment, this invention provides a pharmaceuticallyacceptable tablet comprising ezatiostat hydrochloride, an intragranularexcipient and extragranular excipient wherein the ezatiostathydrochloride comprises from about 75 to about 82 percent by weight ofthe tablet, wherein said tablet is stored in a container with adesiccant.

Crystalline Ansolvate

When used for treating humans, it is important that a crystallinetherapeutic agent like ezatiostat hydrochloride retains its polymorphicand chemical stability, solubility, and other physicochemical propertiesover time and among various manufactured batches of the agent. If thephysicochemical properties vary with time and among batches, theadministration of a therapeutically effective dose becomes problematicand may lead to toxic side effects or to ineffective therapy,particularly if a given polymorph decomposes prior to use, to a lessactive, inactive, or toxic compound. Therefore, it is important tochoose a form of the crystalline agent that is stable, is manufacturedreproducibly, and has physicochemical properties favorable for its useas a therapeutic agent.

It has been discovered that ezatiostat salts and, in particular, thehydrochloride salt, can be formed as a crystalline ansolvate, referredto here as form D. Surprisingly, this ansolvate demonstrates superiorstability and other physicochemical properties compared to the solvatecrystalline forms A, B, C, E, and F. Accordingly, in one aspect, thisinvention provides for tablets comprising crystalline ezatiostatansolvate salt and, in particular, the hydrochloride salt (crystallineform D). In one embodiment, the crystalline ezatiostat hydrochlorideansolvate does not undergo polymorphic transformation. In anotherembodiment, the crystalline ezatiostat hydrochloride ansolvate ischaracterized by an endothermic peak at (177±2)° C. as measured bydifferential scanning calorimetry. In another embodiment, thecrystalline ezatiostat hydrochloride ansolvate is characterized by thesubstantial absence of thermal events at temperatures below theendothermic peak at (177±2)° C. as measured by differential scanningcalorimetry. In another embodiment, the crystalline ezatiostathydrochloride ansolvate is characterized by an X-ray powder diffractionpeak (Cu Kα radiation) at (2.7±0.2)° 2θ. In another embodiment, thecrystalline ezatiostat hydrochloride ansolvate is characterized by anX-ray powder diffraction peak (Cu Kα radiation) at (6.3±0.2)° 2θ. Inanother embodiment, the crystalline ezatiostat hydrochloride ansolvateis characterized by an X-ray powder diffraction pattern (Cu Kαradiation) substantially similar to that of FIG. 3 or FIG. 4. In anotherembodiment, the crystalline ezatiostat hydrochloride ansolvate ischaracterized by a solid-state ¹³C nuclear magnetic resonance spectrumsubstantially similar to that of FIG. 5. In another embodiment, thecrystalline ezatiostat hydrochloride ansolvate is characterized by atleast two X-ray powder diffraction peaks (Cu Kα radiation) selected from2.7°, 6.3°, 7.3°, 8.2°, 8.4°, 9.6°, 11.0°, and 12.7° 2θ (each±0.2° 2θ).In another embodiment, the crystalline ezatiostat hydrochlorideansolvate is characterized by at least three X-ray powder diffractionpeaks (Cu Kα radiation) selected from 2.7°, 6.3°, 7.3°, 8.2°, 8.4°,9.6°, 11.0°, and 12.7° 2θ (each±0.2° 2θ). In another embodiment, thecrystalline ezatiostat hydrochloride ansolvate is characterized by atleast one X-ray powder diffraction peak (Cu Kα radiation) selected from2.7°, 6.3°, 7.3°, 8.2°, 8.4°, 9.6°, 11.0°, and 12.7° 2θ (each±0.2° 2θ).In another embodiment, the crystalline ezatiostat hydrochloride ischaracterized by at least two X-ray powder diffraction peaks (Cu Kαradiation) selected from 2.7°, 6.3°, 7.3°, 8.2°, 8.4°, 9.6°, 11.0°, and12.7° 2θ (each±0.2° 2θ). In another embodiment, the crystallineezatiostat hydrochloride ansolvate is characterized by at least threeX-ray powder diffraction peaks (Cu Kα radiation) selected from 2.7°,6.3°, 7.3°, 8.2°, 8.4°, 9.6°, 11.0°, and 12.7° 2θ (each±0.2° 2θ).

In one embodiment, this invention provides a pharmaceutically acceptabletablet comprising ezatiostat hydrochloride, an intragranular excipient,and extragranular excipient, wherein the ezatiostat hydrochloridecomprises from about 75 to about 82 percent by weight of the tablet andthe ezatiostat hydrochloride comprises crystalline form D. In a furtherembodiment, said tablet contains from about 100 mg to about 1250 mg ofezatiostat hydrochloride.

In another embodiment, this invention provides a pharmaceuticallyacceptable tablet comprising ezatiostat hydrochloride, an intragranularexcipient, and extragranular excipient, wherein the ezatiostathydrochloride comprises from about 75 to about 82 percent by weight ofthe tablet and the ezatiostat hydrochloride comprises crystalline formD, wherein intragranular excipient is selected from one or more ofmannitol, croscarmellose sodium, and hypromellose. In a furtherembodiment, the intragranular excipient comprises a mixture of mannitol,croscarmellose sodium, and hypromellose. In another embodiment, theintragranular excipient comprises from about 17 to about 21 percent byweight of the tablet. In another embodiment, the intragranular excipientcomprises from about 19 to about 20 percent by weight of the tablet.

In another embodiment, this invention provides a pharmaceuticallyacceptable tablet comprising ezatiostat hydrochloride, an intragranularexcipient, and extragranular excipient, wherein the ezatiostathydrochloride comprises from about 75 to about 82 percent by weight ofthe tablet and the ezatiostat hydrochloride comprises crystalline formD, wherein the intragranular excipient comprises mannitol and the amountof mannitol ranges from about 13 to about 15 percent by weight of thetablet.

In another embodiment, this invention provides a pharmaceuticallyacceptable tablet comprising ezatiostat hydrochloride, an intragranularexcipient, and extragranular excipient, wherein the ezatiostathydrochloride comprises from about 75 to about 82 percent by weight ofthe tablet and the ezatiostat hydrochloride comprises crystalline formD, wherein the intragranular excipient comprises croscarmellose sodiumand the amount of croscarmellose sodium ranges from about 1.5 to about3.5 percent by weight of the tablet.

In another embodiment, this invention provides a pharmaceuticallyacceptable tablet comprising ezatiostat hydrochloride, an intragranularexcipient, and extragranular excipient, wherein the ezatiostathydrochloride comprises from about 75 to about 82 percent by weight ofthe tablet and the ezatiostat hydrochloride comprises crystalline formD, wherein the intragranular excipient comprises hypromellose and theamount of hypromellose ranges from about 2 to about 4 percent by weightof the tablet.

In another embodiment, this invention provides a pharmaceuticallyacceptable tablet comprising ezatiostat hydrochloride, an intragranularexcipient, and extragranular excipient, wherein the ezatiostathydrochloride comprises from about 75 to about 82 percent by weight ofthe tablet and the ezatiostat hydrochloride comprises crystalline formD, wherein the tablet comprises an intragranular excipient thatcomprises of mannitol, croscarmellose sodium, and hypromellose, whereinthe amount of mannitol employed in the intragranular excipient mixtureis from about 13.5 to about 14.5 percent by weight of the tablet, theamount of croscarmellose sodium employed in the intragranular excipientmixture is from about 2 to about 3 percent by weight of the tablet, andthe amount of hypromellose employed in the intragranular excipientmixture is from about 2.5 to about 3.5 percent by weight of the tablet.

In another embodiment, this invention provides a pharmaceuticallyacceptable tablet comprising ezatiostat hydrochloride, an intragranularexcipient, and extragranular excipient, wherein the ezatiostathydrochloride comprises from about 75 to about 82 percent by weight ofthe tablet and the ezatiostat hydrochloride comprises crystalline formD, wherein the extragranular excipient comprises one or more ofcroscarmellose sodium and/or magnesium stearate. In a furtherembodiment, the amount of croscarmellose sodium employed in theextragranular excipient mixture is from about 1.5 to about 3.5 percentby weight of the tablet. In another embodiment, the amount of magnesiumstearate employed in the extragranular excipient mixture is from about0.5 to about 1.5 percent by weight of the tablet.

In another embodiment, this invention provides a pharmaceuticallyacceptable tablet comprising ezatiostat hydrochloride, an intragranularexcipient, and extragranular excipient, wherein the ezatiostathydrochloride comprises from about 75 to about 82 percent by weight ofthe tablet and the ezatiostat hydrochloride comprises crystalline formD, wherein the extragranular excipient is selected from one or more ofcroscarmellose sodium and magnesium stearate, wherein the amount ofcroscarmellose sodium employed in the extragranular excipient mixture isfrom about 2 to about 3 percent by weight of the tablet and the amountof magnesium stearate is about 1 percent by weight of the tablet.

In another embodiment, this invention provides a pharmaceuticallyacceptable tablet comprising ezatiostat hydrochloride, an intragranularexcipient and extragranular excipient wherein the ezatiostathydrochloride comprises from about 75 to about 82 percent by weight ofthe tablet and the ezatiostat hydrochloride comprises crystalline formD, wherein said tablet further comprises a film coating.

In another embodiment, this invention provides a pharmaceuticallyacceptable tablet comprising ezatiostat hydrochloride, an intragranularexcipient and extragranular excipient wherein the ezatiostathydrochloride comprises from about 75 to about 82 percent by weight ofthe tablet and the ezatiostat hydrochloride comprises crystalline formD, wherein said tablet comprises about 500 mg of ezatiostathydrochloride. In another embodiment, the tablet comprises preferablyabout 750 mg, more preferably about 1 gm, and even more preferably about1.25 gm of ezatiostat hydrochloride.

In another embodiment, this invention provides a pharmaceuticallyacceptable tablet comprising ezatiostat hydrochloride, an intragranularexcipient and extragranular excipient wherein the ezatiostathydrochloride comprises from about 75 to about 82 percent by weight ofthe tablet and the ezatiostat hydrochloride comprises crystalline formD, wherein said tablet is stored in a container with a desiccant.

In one of its method embodiments, this invention provides a method ofpreparing the solid crystalline ansolvate form D.

In another of its method embodiments, this invention provides a methodof storing crystalline ezatiostat hydrochloride ansolvate such that themorphology of form D remains stable over its shelf-life and, indeed, forprolonged periods of time. In one aspect of this method, the crystallineezatiostat hydrochloride ansolvate in an anhydrous environment (e.g., byusing desiccants or vacuum conditions to maintain an anhydrousenvironment).

Identifying The Ansolvate Form D

A solid form screen was carried out on ezatiostat hydrochloride,starting with ezatiostat hydrochloride monohydrate form A, which waspreviously known. Both thermodynamic and kinetic crystallizationtechniques were employed. Once solid samples were harvested fromcrystallization attempts, they were examined under a microscope forbirefringence and morphology. The solid samples were characterized byvarious techniques including those described above. A number ofdifferent crystallization techniques were used as set forth below.

Fast evaporation: solutions were prepared in various solvents andsonicated between aliquot additions to assist in dissolution. Once amixture reached complete dissolution, as judged by visual observation,the solution was filtered through a 0.2 μm nylon filter. The filteredsolution was allowed to evaporate at room temperature in an open vial,and the solids that formed were isolated by filtration and dried.

Slow evaporation: solutions were prepared as for the fast evaporationtechnique above, and the filtered solution was allowed to evaporate atroom temperature in a vial covered with aluminum foil perforated withpinholes. The solids that formed were isolated by filtration and dried.

Slow cooling: saturated solutions were prepared in various solvents atelevated temperatures and filtered through a 0.2 μm nylon filter into anopen vial while still warm. The vial was covered and allowed to coolslowly to room temperature, and the presence or absence of solids wasnoted. If there were no solids present, or if the amount of solids wasjudged too small for XRPD analysis, the vial was placed in arefrigerator overnight. Again, the presence or absence of solids wasnoted and if there were none, the vial was placed in a freezerovernight. Solids that formed were isolated by filtration and dried.

Crash cooling: saturated solutions were prepared in various solvents orsolvent systems at an elevated temperature and filtered through a 0.2-μmnylon filter into an open vial while still warm. The vial was coveredand placed directly into a freezer. The presence or absence of solidswas noted. Solids that formed were isolated by filtration and dried.

Antisolvent crystallization: solutions were prepared in various solventsat elevated temperature and filtered through a 0.2-μm nylon filter.Solid formation was induced by adding the filtered solution to anappropriate anti-solvent at a temperature below room temperature. Theresulting solids were isolated by filtration and dried.

Slurrying: slurries were prepared by adding enough solids to a givensolvent so that undissolved solids were present. The mixture was thenagitated in a sealed vial at a chosen temperature. After time, thesolids were isolated by filtration and dried.

Stress experiments: solids were stressed under different temperatureand/or relative humidity (RH) environments for a measured time period.Specific RH values were achieved by placing the sample inside sealedchambers containing saturated salt solutions. Samples were analyzed byXRPD immediately after removal from the stress environment.

In addition to the starting material identified as form A, fiveadditional solid forms were identified. Of the five additional forms,only one, form D, was confirmed to have an unsolvated structure,crystalline ezatiostat hydrochloride ansolvate. The other four formswere determined to be either hydrates, other solvates, or unstableforms.

Ansolvate Form D and its Properties

In one embodiment, this invention provides a crystalline ezatiostat saltansolvate and, in particular, the hydrochloride ansolvate (crystallineform D). In another embodiment, this invention provides a compositioncomprising the crystalline ezatiostat hydrochloride ansolvate.Preferably, the crystalline form D is substantially free of a solvatedpolymorph of ezatiostat hydrochloride. “Substantially free” of asolvated polymorph of ezatiostat hydrochloride refers to a crystallineform D, which excludes solvated polymorph of ezatiostat hydrochloride toan extent that the form D crystals are suitable for humanadministration. In one embodiment, the crystalline form D contains up toabout 5%, more preferably about 3%, and still more preferably about 1%of one or more solvated polymorph of ezatiostat hydrochloride. As usedherein, solvate includes hydrate form as well.

It is possible to attain the ansolvate form D with such high polymorphicpurity due, in part, to the surprising stability of the ansolvate, andits resistance to conversion to a solvate form, even when stored at 40°C. and 75% RH without a desiccant for 6 months. See Table 5 below. Incontrast, the solvate form E transforms almost entirely to form Bcrystals merely during tablet manufacture, which then transforms into amixture of form B and the ansolvate form D within 3 months of storage at40° C. and 75% RH without a desiccant. See Table 6 below. The solvateform A is also polymorphically unstable, converting into a mixture offorms A and D during manufacture. See Table 7 below.

Not only was the ansolvate form D polymorphically stable, it was alsomore stable to chemical degradation compared to the polymorphs A, B, andE. See Tables 5-7 below in rows entitled “Total impurities.” Polymorphicform B, obtained from form E during tablet manufacture, was the mostunstable, decomposing at more than double the rate of decomposition ofthe ansolvate form D. The stability of form D was enhanced even more,when stored in presence of a desiccant. Thus, in another embodiment, thepresent invention provides a crystalline ansolvate form D, which, whenexposed to a temperature of about 25° C. for up to about 6 months in thepresence of a desiccant, does not show substantial formation of animpurity. As used herein, “in the presence of a desiccant” refers to thedesiccant being placed in a closed container with the ansolvate form D.The closed container, may be, but need not be sealed such that the airfrom the surrounding can not enter the closed container.

As used herein, “impurity” refers to one or more of: TLK 236, anotherpolymorphic form of ezatiostat hydrochloride including withoutlimitation form A, B, C, E, or F, and any other compound other thanezatiostat hydrochloride ansolvate, which may be identified by HPLC. TLK236 is a monoester derived from the partial hydrolysis of ezatiostatwhere the phenyl glycine moiety remains esterified. “Does not showsubstantial formation of an impurity” refers to formation of only up toabout 1.5% or more preferably up to about 1% of impurity.

The crystal form D is desirable from yet another standpoint, which isthat, surprisingly, no other ansolvate form being identified uponscreening, the ansolvate form D can not convert to another ansolvatepolymorph upon storage or handling. And, as described above, ansolvateform D is stable with respect to a conversion to a solvate form, such asA, B, or E.

In another aspect, the present invention provides a method of storingcomprising storing the crystalline ezatiostat hydrochloride ansolvateform D in the presence of a desiccant. In one embodiment, the desiccantis amorphous silicate. In another embodiment, the desiccant is Sorb-Itsilica gel. In one embodiment, the ansolvate form D is stored for up to3 months, up to 6 months, up to 9 months, up to 1 year, up to 1.5 years,up to 2 years, up to 3 years, or up to 4 years. In another embodiment,the ansolvate form D is stored at a temperature of up to about 5° C. Inanother embodiment, the ansolvate form D is stored at a temperature ofup to about 25° C. In another embodiment, the ansolvate form D is storedat a temperature of up to about 40° C.

Furthermore, as part of a tablet, the ansolvate form D demonstratedhigher aqueous dissolution rate than polymorphic form E (which convertsto form B upon tableting) or B, when measured in 0.1 molar HCl, which isa convenient model for gastric fluid. Without being bound by theory, ahigher dissolution rate relates to a higher amount of the active agentin the gastric fluid, which in turn relates to higher bioavailability ofthe active agent. A high bioavailability is desired, for example andwithout limitation, for reducing inter patient variability of drugexposure for a orally administered agent such as ezatiostathydrochloride. So, for therapeutic use, the ansolvate form D iscontemplated to be advantageous over form B or E. In one embodiment, thepresent invention provides a composition including the crystalline formD, which shows an aqueous solubility of at least about 5 mg/mL to about20 mg/mL, about 10 mg/mL to about 15 mg/mL, about 5 mg/mL to about 15mg/mL, or about 15 mg/mL to about 20 mg/mL. The aqueous solubility canbe measured in a variety of aqueous solvents, including withoutlimitation, water, 0.9% aqueous NaCl, 5% dextrose for injection,phosphate buffered saline, and generally aqueous solutions having a pHof less than about 5. Such solvents may include suitable buffers andother salts.

Preparation of Ansolvate Form D

In another aspect, this invention provides a method of preparing thesolid crystalline ansolvate provided herein. In one embodiment, themethod comprises slurrying ezatiostat hydrochloride in methyl tert-butylether at room temperature. In another embodiment, the method comprisesslurrying ezatiostat hydrochloride in hexanes at about 60° C. In anotherembodiment, the method comprises heating ezatiostat hydrochloridemonohydrate form A at a temperature from above about 155° C. up to lessthan the decomposition temperature and preferably to no more than about180° C. for a period sufficient to convert the monohydrate to theansolvate form D. Based on the present disclosure such transformationscan be readily performed by the skilled artisan, for example, bymonitoring DSC results.

In still another aspect, ezatiostat hydrochloride ansolvate is alsoobtained by dissolution of crude hydrated ezatiostat hydrochloride inabout 5.6 times its weight of ethanol, heating to about (65-70)° C.,filtering, seeding with a small quantity (e.g. about 2% by weight of theinitial ezatiostat hydrochloride) of ezatiostat hydrochloride ansolvate,cooling to about 40° C., adding ethyl acetate in about 13.5 times theweight of the ezatiostat hydrochloride ansolvate, gradually cooling toabout (20-25)° C. and then to (−5-0)° C., then filtering, washing withethyl acetate, and drying.

Characterization of Crystalline Forms of Ezatiostat Hydrochloride

Crystalline ezatiostat hydrochloride ansolvate is characterized by itschemical composition, i.e. the presence of ezatiostat hydrochloride andthe absence of water or other solvents of crystallization, and thecrystalline nature of the material (the presence of an XRPD patterncharacteristic of a crystalline, as opposed to amorphous, material). Itmay further conveniently be characterized by methods such as DSC, XRPD,and SS-NMR. It may also be characterized by other methods. These includeanalysis for water determination (typically by Karl Fischer analysis),where none or only a small quantity of water—significantly less thanthat which would be expected from a hydrate such as themonohydrate—should be found; and TG or TG-IR analysis, where none oronly a small weight loss—significantly less than that which would beexpected by the loss of a solvent of crystallization—would be found.

By DSC, crystalline ezatiostat hydrochloride ansolvate is characterizedby an endothermic peak at (177±2)° C., which corresponds to melting ofthe crystalline ezatiostat hydrochloride ansolvate. If the crystallineezatiostat hydrochloride ansolvate is free of other forms of ezatiostathydrochloride, the DSC pattern will be characterized also by thesubstantial absence of thermal events at temperatures below theendothermic peak at (177±2)° C.; but the presence of minor quantities ofother forms such as ezatiostat hydrochloride monohydrate will result inthe presence of minor thermal events at lower temperatures. As usedherein, “substantial absence of thermal events” refer to endotherms andexotherms related to melting and recrystallization.

In one embodiment, this invention provides a tablet comprising acrystalline ansolvate form D characterized by an endothermic peak at(177±2)° C. as measured by differential scanning calorimetry (DSC). Inanother embodiment, this invention provides a tablet comprising acrystalline ansolvate form D characterized by substantial absence ofthermal events at temperatures below the endothermic peak at (177±2)° C.as measured by differential scanning calorimetry. See, FIG. 6, whichgraphically illustrates a comparative DSC of forms A, D, and E, anddemonstrates substantial absence of thermal events at temperatures belowthe endothermic peak at (177±2)° C. for the crystalline ansolvate formD.

Under XRPD, crystalline ezatiostat hydrochloride ansolvate ischaracterized by a dominant zone with a rectangular planar(2-dimensional) unit cell with axial lengths of about 18.28 Å and 64.23Å and an included angle of 90°; and systematic extinctions indicatingthat the planar cell has p2gg symmetry. Only two 3-dimensional spacegroups are consistent with the observed dominant zone cell and anordered packing of a single diastereomer of a chiral molecule: these areorthorhombic space groups (P2₁2₁2 or P2₁2₁2₁) with approximate unit celldimensions of a=64.23 Å, b=18.28 Å, c=short (P2₁2₁2), or a=short,b=18.28 Å, c=64.23 Å (P2₁2₁2₁). Note that permutations of the a and baxes are permissible for P2₁2₁2, and of all three axes for P2₁2₁2₁. Thelowest-angle feature not related to the dominant zone is near 17.5° 2θ,indicating a short axis of about 5.1 Å (best match indexing solutionsare consistent with about 5.08 Å, but there is insufficient peakresolution above 17° 2θ to definitively determine the length of theshort axis and the space group). XRPD patterns will show peakscharacteristic of this unit cell, as discussed further in the Examplesbelow.

In another embodiment, this invention provides a tablet comprising acrystalline ansolvate form D characterized by at least one X-ray powderdiffraction peak (Cu Kα radiation) selected from 2.7°, 6.3°, 7.3°, 8.2°,8.4°, 9.6°, 11.0°, and 12.7° 2θ (each±0.2° 2θ). In another embodiment,this invention provides a crystalline ansolvate form D characterized byan X-ray powder diffraction peak (Cu Kα radiation) at (2.7±0.2)° 2θ. Inanother embodiment, this invention provides a tablet comprising acrystalline ansolvate form D characterized by an X-ray powderdiffraction peak (Cu Kα radiation) at (6.3±0.2)° 2θ. In anotherembodiment, this invention provides a tablet comprising a crystallineansolvate form D characterized by at least two X-ray powder diffractionpeaks (Cu Kα radiation) selected from 2.7°, 6.3°, 7.3°, 8.2°, 8.4°,9.6°, 11.0°, and 12.7° 2θ (each±0.2° 2θ). In another embodiment, thisinvention provides a tablet comprising a crystalline ansolvate form Dcharacterized by at least three X-ray powder diffraction peaks (Cu Kαradiation) selected from 2.7°, 6.3°, 7.3°, 8.2°, 8.4°, 9.6°, 11.0°, and12.7° 2θ (each±0.2° 2θ). In another embodiment, this invention providesa tablet comprising a crystalline ansolvate form D characterized by atleast one X-ray powder diffraction peak (Cu Kα radiation) selected from2.7°, 6.3°, 7.3°, 8.2°, 8.4°, 9.6°, 11.0°, and 12.7° 2θ (each±0.2° 2θ).

In another embodiment, this invention provides a tablet comprising acrystalline ansolvate form D characterized by an X-ray powderdiffraction pattern (Cu Kα radiation) substantially similar to that ofFIG. 3 or FIG. 4. In another embodiment, this invention provides atablet comprising a crystalline ansolvate form D characterized by asolid-state ¹³C nuclear magnetic resonance spectrum substantiallysimilar to that of FIG. 5.

Methods of therapeutic uses of ezatiostat are disclosed in U.S.Provisional Patent Applications 61/352,371, 61/352,373, and 61/352,374,each of which was filed on Jun. 7, 2010; the contents of which areincorporated herein by reference in their entirety.

It will be apparent to those skilled in the art that many modificationsof the above examples, both to materials and methods, may be practicedwithout departing from the scope of the current invention.

EXAMPLES

The following examples describe the preparation of a tablet comprisingezatiostat hydrochloride, as well as the preparation, characterization,and properties of ezatiostat hydrochloride ansolvate. Unless otherwisestated, all temperatures are in degrees Celcius (° C.) and the followingabbreviations have the following definitions:

-   -   DSC=Differential scanning calorimetry    -   NA=Not applicable    -   Q=Percent dissolved per unit time    -   RH=Relative humidity    -   RSD=Relative standard deviation    -   RRT=Relative retention time    -   SS-NMR=Solid state nuclear magnetic resonance    -   TG-IR=Thermogravimetric infra red analysis    -   XRPD=X-ray powder diffraction    -   VT-XRPD=Variable temperature X-ray powder diffraction

Example 1 Formulations of Ezatiostat Hydrochloride

Two different formulations comprising ezatiostat hydrochloride wereprepared by mixing ezatiostat hydrochloride with the each of theexcipient mixtures 1 and 2 in a 3.3:1 ratio. Specifically, formulation 1was prepared by mixing 75 mg of excipient mixture 1 with 250 mg ofezatiostat hydrochloride, whereas formulation 2 was prepared by mixing75 mg of excipient mixture 2 with 250 mg of ezatiostat hydrochloride.Table 3 provides the different ingredients used in the excipientmixtures 1 and 2.

TABLE 3 Excipient mixtures for ezatiostat hydrochloride formulationsExcipient mixture Excipient mixture Ingredient 1 (mg) 2 (mg)Microcrystalline cellulose 88.25 (Avicel PH 112) Mannitol granular 88.25(Mannogem granular 2080) Croscarmellose sodium 32.5 Crospovidone 32.5Providone K-29/32 19.5 HPMC E5 Premium 19.5 Colloidal silicon dioxide3.25 3.25 Magnesium stearate 6.5 6.5

Example 2 General Process for Preparing a Tablet Comprising EzatiostatHydrochloride

The required amounts of Ezatiostat hydrochloride, mannitol,croscarmellose sodium and hypromellose were dispensed and milled asnecessary. The mixture was then dry blended in a high shear granulator.Adequate amount of purified water was added and the mixture was thengranulated. The granulate was wet screened and dried in a fluid beddryer. The dried granules were milled and the granulate was then blendedwith adequate amount of croscarmellose sodium in a V blender. Then themixture was blended with magnesium stearate in a V blender. The blendwas compressed into tablets and coated with an Opadry® Clear solution.The coated tablets were dispensed into bottles, induction seal wasapplied, and the bottles were capped and labeled. Table 4 shows thevarious parameters for four different lots of the tablet.

TABLE 4 Compression Parameters for the tablet Drug Product Lot NumberParameter 1 2 3 4 Compression 7.1 15.5 13.2 13.5 pressure (kN) TabletWeight Mean 654.2 652.5 655.8 653.2 Target: Range 624-681 645-659651-665 648-660 650 mg Mean 654.0 653.2 651.1 651.5 Range: Range 628-682650-657 648-655 648-655 618-683 mg Mean 652.2 653.2 651.8 651.2 Range635-667 649-657 647-657 648-654 Tablet Mean 11.1 11.5 12.1 13.0 HardnessRange  8.3-15.3 10.6-12.0 10.7-13.8 11.0-14.3 Target: Mean 11.3 10.610.5 11.4 12 kP Range  9.3-12.5  9.6-11.0  9.8-11.3 10.5-12.5 Range:Mean 11.4 10.5 11.3 12.3 8-16 kP Range  9.2-13.6 10.2-11.0 10.6-12.210.5-13.3 Tablet Mean 6.47 5.95 6.04 6.08 Thickness Range 6.32-6.615.92-6.05 6.00-6.07 6.04-6.20 Target: Mean 6.45 5.95 6.02 6.09 6.50 mmRange 6.39-6.53 5.94-5.97 5.99-6.10 6.07-6.11 Range: Mean 6.41 5.95 5.966.01 5.50-7.00 mm Range 6.38-6.50 5.92-6.00 5.94-5.99 5.99-6.02Friability Begin. 0.2% 0.2% 0.2% 0.2% Spec: End 0.2% 0.2% 0.3% 0.2% NMT0.8% Disinte- Begin. 0:02:34 0:09:53 0:08:23 0:07:15 gration Spec.: End0:02:48 0:10:50 0:11:46 0:07:48 NMT 15 min.

Example 3 Preparation of Ezatiostat Hydrochloride Ansolvate by Slurrying

Ezatiostat hydrochloride monohydrate was added to methyl tert-butylether at room temperature in excess, so that undissolved solids werepresent. The mixture was then agitated in a sealed vial at roomtemperature for 4 days, and the solids were then isolated by suctionfiltration. XRPD analysis of the solids established that the isolatedsolids were ezatiostat hydrochloride ansolvate.

Ezatiostat hydrochloride monohydrate was added to hexanes at 60° C. inexcess, so that undissolved solids were present. The mixture was thenagitated in a sealed vial at 60° C. for 4 days, and the solids were thenisolated by suction filtration. XRPD analysis of the solids establishedthat the isolated solids were ezatiostat hydrochloride ansolvate.

Example 4 Preparation of Crystalline Ezatiostat Hydrochloride Ansolvateby Heating

DSC of crystalline ezatiostat hydrochloride monohydrate showed thepattern in FIG. 1, as discussed in paragraph above. Hot stage microscopyshowed an initial melt followed by a recrystallization at 153° C. and afinal melt at 166° C. VT-XRPD, where XRPD patterns were obtained at 28°C., 90° C., and 160° C. during heating, and 28° C. after cooling of theformerly heated material, showed the presence of ezatiostathydrochloride monohydrate at 28° C. and 90° C. during heating and ofcrystalline ezatiostat hydrochloride ansolvate at 160° C. and 28° C.after cooling of the formerly heated material. This confirmed that thetransition at around 153/156° C. was a conversion of ezatiostathydrochloride monohydrate form A to crystalline ezatiostat hydrochlorideansolvate form D and that the final DSC endothermic peak at about 177°C. (166° C. in the hot stage microscopy) was due to the melting ofcrystalline ezatiostat hydrochloride ansolvate. This was furtherconfirmed by XRPD of the TG-IR material, where XRPD patterns obtained atroom temperature both before and after heating to about 160° C. showedthat the material before heating was form A and that the material afterheating was form D ansolvate. DSC of crystalline ezatiostathydrochloride ansolvate prepared by recrystallization showed the patternin FIG. 2, with only the endothermic peak at about 177° C. followed by abroad endotherm at about (205-215)° C. Accordingly, the presence of theDSC endothermic peak at about 177° C., for example at (177±2)° C., whenmeasured under the conditions described above, is consideredcharacteristic of crystalline ezatiostat hydrochloride ansolvate, andthe substantial absence of thermal events at temperatures below this isconsidered indicative of the absence of other forms of ezatiostathydrochloride.

Example 5 Preparation of Crystalline Ezatiostat Hydrochloride Ansolvateby Crystallization

61.5 Kg crude ezatiostat hydrochloride was added to a reactor at roomtemperature, followed by 399 liter (L) ethanol, and this mixture washeated to 68° C. to completely dissolve the ezatiostat hydrochloride,filtered, then allowed to cool to 65° C. and checked for clarity and theabsence of crystallization. About 1.3 Kg of ezatiostat hydrochlorideansolvate form D was suspended in 9 L of ethyl acetate, and aboutone-half of this suspension was added to the ethanol solution. Themixture was cooled to 63° C. and the second half of the suspension addedto the mixture. The resulting mixture was cooled gradually to 45° C.,928 L ethyl acetate was added, and the mixture was cooled to 26° C. andheld at about that temperature for about 5 hours, then cooled to −2° C.The mixture, containing crystalline ezatiostat hydrochloride ansolvate,was filtered, and the residue washed twice with 65 L of chilled (0-5°C.) ethyl acetate. The crystalline ezatiostat hydrochloride ansolvatewas dried at 30° C. for 48 hours, then cooled to room temperature andsieved. Analysis of the material by DSC and XRPD confirmed its identityas crystalline ezatiostat hydrochloride ansolvate, and Karl Fischeranalysis showed a water content of 0.1%.

XRPD of form D showed the pattern in FIG. 3. High-resolution XRPD ofform D showed the pattern in FIG. 4. The major peaks are at 2.7°, 5.0°,5.5°, 6.3°, 7.3°, 8.2°, 8.4°, 9.6°, 10.1°, 11.0°, 12.0°, 12.7°, 13.3°,13.8°, 14.8°, 15.1°, 15.6°, 16.1°, 16.6°, 17.3°, 17.5°, 17.8°, 18.0°,18.4°, 18.7°, 19.0°, 19.5°, 20.0°, 20.5°, 21.3°, 21.7°, 22.1°, 22.3°,23.0°, 23.2°, 23.5°, 23.8°, 24.4°, 24.9°, 25.4°, 25.7°, 26.4°, 26.7°,27.2°, 27.6°, 27.8°, 28.0°, and 29.3° 2θ. These peaks listed here atless than about 15° 2θ exhibit good separation from each other and areeasily discernable even at lower resolution. Low angle peaks such as thepeaks at 2.7°, 6.3°, 7.3°, 8.2°, 8.4°, 9.6°, 11.0°, and 12.7° 2θ areparticularly useful in characterization of crystalline ezatiostathydrochloride ansolvate; and at least one, preferably at least two, morepreferably at least three of these peaks may be used. In particular, thepeaks at 2.7° and 7.3° 2θ, especially the peak at 2.7° 2θ, may beconsidered characteristic of crystalline ezatiostat hydrochlorideansolvate.

SS-NMR analysis of crystalline ezatiostat hydrochloride ansolvate showedthe pattern in FIG. 5, clearly distinguishable from that of ezatiostathydrochloride monohydrate.

In summary, crystalline ezatiostat hydrochloride ansolvate form D ischaracterized by chemical composition, i.e. the presence of ezatiostathydrochloride and the absence of water or other solvents ofcrystallization, and the crystalline nature of the material (thepresence of an XRPD pattern characteristic of a crystalline, as opposedto amorphous, material). Additionally, the presence of the DSCendothermic peak at (177±2)° C. alone, or the presence of one or more ofthe low angle XRPD peaks (especially the peak at 2.7° 2θ, alone or withone or more of the other peaks below 15° 2θ, such as the peaks at 6.3°,7.3°, 8.2°, 8.4°, 9.6°, 11.0°, and 12.7° 2θ, especially such as the peakat 7.3° 2θ and optionally one or more of the other peaks listed),preferably also in the absence of peaks indicative of ezatiostathydrochloride monohydrate or other forms of ezatiostat hydrochloride,are considered characteristic of crystalline ezatiostat hydrochlorideansolvate. Also considered characteristic of crystalline ezatiostathydrochloride ansolvate is XRPD patterns substantially the same as thosein FIG. 3 or FIG. 4, when measured under the conditions described above.

Example 6 Polymorphic and Physicochemical Stability of Form D Ansolvatein the Absence of Desiccants

This example demonstrates the superior stability and solubility of theansolvate form D compared to the solvate forms A, B, and E. Tablets offorms A, D, and E were made and stored at 40° C./75% RH without adesiccant for up to 6 months and the various properties of the tabletsdetermined initially, and at 3 and 6 month intervals. As describedabove, form E converts to form B simply during tableting. The resultsare tabulated below.

TABLE 5 Polymorphic and Physicochemical Stability of Form D Ansolvate inthe Absence of Desiccants when stored at 40° C./75% RH API PolymorphForm Polymorph Form D Timepoint Initial 3 Month 6 Month DescriptionWhite to off- White round tablet Off-white round Brown round tabletwhite round tablet tablet Assay (HPLC) 93.0-107.0% 101.4 100.3 96.5Label Claim Dissolution Q = 70% of At 45 min, At 45 min, At 45 min,label claim individual results: individual results: individual results:dissolved in 37, 50, 32, 73, 95, 93, 96, 67, 68, 95, 73, 80, 45 min 54,57 98, 81 71, 100 Mean = 51 Mean = 88 Mean = 81 RSD % = 29.1 RSD % =13.6 RSD % = 16.3 Water Content ≦5.0% 0.9 0.8 0.8 X-ray Report PolymorphD Polymorph D Polymorph D Diffraction results Individual RRT = 0.59/0.62ND ND 0.08 Impurities RRT = 0.74 0.21 0.21 0.20 RRT = 0.80 ND ND ND RRT= 0.81 ND ND ND RRT = 0.83 ND 0.07 0.09 RRT = 0.86 ND ND ND TLK 236 RRT= 0.88 0.35 1.33 2.07 RRT = 0.94 ND 0.07 ND RRT = 0.96 0.18 0.19 0.23RRT = 0.99 ND ND ND Total 0.7 1.9 2.7 impurities

TABLE 6 Polymorphic and Physicochemical Stability of Form E in theAbsence of Desiccants when stored at 40° C./75% RH API Polymorph FormPolymorph Form E Timepoint Initial 3 Month 6 Month Description White tooff- White round tablet Off-white Brown round tablet white round roundtablet tablet Assay (HPLC) 93.0-107.0% 93.7 90.3 84.6 Label ClaimDissolution Q = 70% of At 45 min, At 45 min, At 45 min, label claimindividual results: individual results: individual results: dissolved in47, 49, 43, 45, 47, 31, 27, 30, 17, 18, 18, 21, 45 min 47, 47 23, 42 26,16 Mean = 46 Mean = 33 Mean = 19 RSD % = 4.3 RSD % = 26.9 RSD % = 19.4Water Content ≦5.0% 3.5 2.3 2.3 X-ray Report results Polymorph BPolymorph B and D Polymorph B and D Diffraction Individual RRT =0.59/0.62 ND 0.07 0.15 Impurities RRT = 0.74 0.38 0.42 0.51 RRT = 0.80ND 0.16 0.41 RRT = 0.81 ND 0.14 0.17 RRT = 0.83 0.34 0.31 0.16 RRT =0.86 ND 0.06 ND TLK 236 RRT = 0.88 0.42 3.45 4.66 RRT = 0.94 ND 0.08 NDRRT = 0.96 0.20 0.19 0.24 RRT = 0.99 0.12 0.20 ND Total 1.5 5.1 6.3impurities

TABLE 7 Polymorphic and Physicochemical Stability of Form A in theAbsence of Desiccants when stored at 40° C./75% RH API Polymorph FormPolymorph Form A Timepoint Initial 3 Month 6 Month Description White tooff- White round tablet Off-white round Off-white round white roundtablet tablet tablet Assay (HPLC) 93.0-107.0% 97.2 94.1 91.5 Label ClaimDissolution Q = 70% of At 45 min, At 45 min, At 45 min, label claimindividual results: individual results: individual results: dissolved12, 12, 11, 12, 88, 49, 64, 81, 79, 83, 73, 80, in 45 min 12, 11 77, 8325, 86 Mean = 12 Mean = 74 Mean = 71 RSD % = 4.0 RSD % = 19.7 RSD % =32.5 Water Content ≦5.0% 2.1 1.7 1.9 X-ray Report results Polymorph Aand D Polymorph A and D Polymorph A and D Diffraction Individual RRT =0.59/0.62 ND ND 0.07 Impurities RRT = 0.74 0.13 0.16 0.15 RRT = 0.80 ND0.08 0.14 RRT = 0.81 ND 0.07 0.05 RRT = 0.83 0.46 0.10 ND RRT = 0.86 NDND ND TLK 236 RRT = 0.88 0.45 1.99 2.92 RRT = 0.94 ND 0.07 ND RRT = 0.960.16 0.16 0.21 RRT = 0.99 ND 0.07 ND Total 1.2 2.7 3.5 impurities

Example 7 Polymorphic and Physicochemical Stability of Form D Ansolvatein Presence of Desiccants

The stability of the ansolvate form D was further improved when storedin presence of a desiccant as demonstrated in this example. Tablets ofansolvate form D, were packaged with and without desiccant (Sorb-ItCannister, 1 gram). Fifty tablets were packaged in a round, white 1500mL bottle with a screw cap over an induction seal. Impurities wereassayed by HPLC. When stored at 25° C./60% RH with desiccant for 3months, no increase in total impurities was observed. When stored at 40°C./75% RH with desiccant for 3 months, total impurities increased onlyby 0.3%. When stored at 40° C./75% RH without desiccant for 3 months,total impurities still increased by 1.1%. As tabulated below, thepresence of desiccant appears to further increase the stability of theansolvate form D. Even though the dissolution rate of tablets containingpolymorph D from one batch was below specification (<70%) at an initialtest as well as after 3 months at 25° C./60% RH with desiccant, thesubsequent batches met the required specification.

TABLE 8 Polymorphic and Physicochemical Stability of Form D Ansolvate inPresence of Desiccants Timepoint Initial 3 Month 3 Month 3 Month Storage40° C./75% RH 25° C./60% 40° C./75% without RH with RH with NA desiccantdesiccant desiccant Description White to off- White round Off-whiteround White round Off-white white round tablet tablet tablet roundtablet tablet Assay 93.0-107.0% 101.4 100.3 99.7 100.8 (HPLC) LabelClaim Dissolution Q = 70% of At 45 min, At 45 min, At 45 min, At 45 min,label claim individual results: individual results: individual results:individual results: dissolved in 37, 50, 32, 73, 95, 93, 96, 67, 36, 57,43, 29, 87, 52, 84, 97, 45 min 54, 57 98, 81 53, 59 77, 59 Mean = 51Mean = 88 Mean = 46 Mean = 76 RSD % = 29.1 RSD % = 12.0 RSD % = 12.2 RSD% = 17.1 Water Content ≦5.0% 0.9 0.8 0.5 0.6 X-ray Report resultsPolymorph D Polymorph D Polymorph D Polymorph D Diffraction IndividualRRT = 0.21 0.18 0.16 0.16 Impurities 0.74/0.72 RRT = 0.83 ND 0.08 ND0.09 TLK 236 RRT = 0.88 0.35 1.3 0.34 0.53 RRT = 0.94 ND 0.07 ND 0.06RRT = 0.96 0.18 0.18 0.19 0.19 Total 0.7 1.8 0.7 1.0 impurities

While this invention has been described in conjunction with specificembodiments and examples, it will be apparent to a person of ordinaryskill in the art, having regard to that skill and this disclosure, thatequivalents of the specifically disclosed materials and methods willalso be applicable to this invention; and such equivalents are intendedto be included within the following claims.

1. A pharmaceutically acceptable tablet comprising ezatiostathydrochloride, an intragranular excipient, and an extragranularexcipient, wherein the ezatiostat hydrochloride comprises from about 75to about 82 percent by weight of the tablet.
 2. The pharmaceuticallyacceptable tablet of claim 1, wherein the ezatiostat hydrochloridecomprises crystalline form D.
 3. The pharmaceutically acceptable tabletof claim 1, wherein said tablet comprises from about 100 mg to about1250 mg of ezatiostat hydrochloride.
 4. The pharmaceutically acceptabletablet of claim 1, wherein the intragranular excipient is selected fromthe group consisting of mannitol, croscarmellose sodium, andhypromellose.
 5. The pharmaceutically acceptable tablet of claim 4,wherein the intragranular excipient comprises a mixture of mannitol,croscarmellose sodium, and hypromellose.
 6. The pharmaceuticallyacceptable tablet of claim 5, wherein the intragranular excipientcomprises from about 17 to about 21 percent by weight of the tablet. 7.The pharmaceutically acceptable tablet of claim 6, wherein total amountof the intragranular excipient is from about 19 to about 20 percent byweight of the tablet.
 8. The pharmaceutically acceptable tablet of claim7, wherein the amount of mannitol employed in the intragranularexcipient mixture ranges from about 13 to about 15 percent by weight ofthe tablet.
 9. The pharmaceutically acceptable tablet of claim 7,wherein the amount of croscarmellose sodium employed in theintragranular excipient mixture ranges from about 1.5 to about 3.5percent by weight of the tablet.
 10. The pharmaceutically acceptabletablet of claim 7, wherein the amount of hypromellose employed in theintragranular excipient mixture ranges from about 2 to about 4 percentby weight of the tablet.
 11. The pharmaceutically acceptable tablet ofclaim 1, wherein the amount of mannitol employed in the intragranularexcipient mixture is from about 13.5 to about 14.5 percent by weight ofthe tablet, the amount of croscarmellose sodium employed in theintragranular excipient mixture is from about 2 to about 3 percent byweight of the tablet, and the amount of hypromellose employed in theintragranular excipient mixture is from about 2.5 to about 3.5 percentby weight of the tablet.
 12. The pharmaceutically acceptable table ofclaim 1, wherein the extragranular excipient is selected from one ormore of croscarmellose sodium and magnesium stearate.
 13. Thepharmaceutically acceptable tablet of claim 12, wherein the amount ofcroscarmellose sodium employed in the extragranular excipient mixture isfrom about 1.5 to about 3.5 percent by weight of the tablet.
 14. Thepharmaceutically acceptable tablet of claim 12, wherein the amount ofmagnesium stearate employed in the extragranular excipient mixture isfrom about 0.5 to about 1.5 percent by weight of the tablet.
 15. Thepharmaceutically acceptable tablet of claim 12, wherein the amount ofcroscarmellose sodium employed in the extragranular excipient mixture isfrom about 2 to about 3 percent by weight of the tablet and the amountof magnesium stearate is about 1 percent by weight of the tablet. 16.The pharmaceutically acceptable tablet of claim 1, wherein the amount ofmannitol employed in the intragranular excipient mixture is from about13.5 to about 14.5 percent by weight of the tablet, the amount ofcroscarmellose sodium employed in the intragranular excipient mixture isfrom about 2 to about 3 percent by weight of the tablet, and the amountof hypromellose employed in the intragranular excipient mixture is fromabout 2.5 to about 3.5 percent by weight of the tablet; and furtherwherein the amount of croscarmellose sodium employed in theextragranular excipient mixture ranges from about 2 to about 3 percentby weight of the tablet and the amount of magnesium stearate employed inthe extragranular mixture is about 1 percent by weight of the tablet.17. The pharmaceutically acceptable tablet according to claim 1, whereinsaid tablet further comprises a film coating.
 18. The pharmaceuticallyacceptable tablet according to claim 1, wherein said tablet comprisesabout 500 mg of ezatiostat hydrochloride.
 19. The pharmaceuticallyacceptable tablet according to claim 1, wherein said tablet comprisesabout 750 mg of ezatiostat hydrochloride.
 20. The pharmaceuticallyacceptable tablet according to claim 1, wherein said tablet comprisesabout 1 g of ezatiostat hydrochloride.
 21. The pharmaceuticallyacceptable tablet according to claim 1, wherein said tablet comprisesabout 1.25 g of ezatiostat hydrochloride.
 22. The pharmaceuticallyacceptable table according to claim 1, wherein said tablet is stored ina container with a desiccant.